Tge virus vaccine

ABSTRACT

ORAL DOSAGE FORM OF VILLIARLY EFFECTIVE BIOLOGICALS FOR ADMINISTRATION TO ANIMALS COMPRISES ENTERIC-COATED PARTICLES HAVING CONTROLLED MAXIMUM SIZE. VACCINE FOR PROTECTION OF SWINE AGAINST TGE COMPRISES ENTERICALLY COATED PARTICLES, LESS THAN ABOUT 2.0 MM. IN DIAMETER, CONTAINING ATTENUATED TGE VIRUS VACCINE.

"United States Patent 3,823,228 TGE VIRUS VACCINE Deam H. Ferris,Greenport, N.Y., and Angel S. Arambulo, Chicago, Ill., assiguors toUniversity of Illinois Foundation, Urbana, 111.

No Drawing. Continuation-impart of abandoned application Ser. No.184,910, Sept. 29, 1971. This application Aug. 30, 1972, Ser. No.285,036

Int. Cl. A61j 3/07; C12k 1/08 US. Cl. 424-35 Claims ABSTRACT OF THEDISCLOSURE Oral dosage form of villiarly effective biologicals foradministration to animals comprises enteric-coated particles havingcontrolled maximum size. Vaccine for protection of swine against TGEcomprises enterically coated particles, less than about 2.0 mm. indiameter, containing attenuated TGE virus vaccine.

This application is a continuation-in-part of our copending applicationSer. No. 184,910, filed Sept. 29, 1971, now abandoned.

This invention relates generally to the oral administration ofbiologicals to animals. More particularly, it relates to a novel dosageform of oral vaccine for protection of swine against TGE and to themethod of protecting swine by administering the vaccine.

Transmissible gastroenteritis, commonly called TGE, is a highlycontagious viral disease which attacks swine of all ages. While thedisease is severe in all swine it is particularly acute in baby pigs, inwhich it causes severe vomiting, diarrhea, and weight loss withconsequent high mortality rates. The disease has been known for manyyears but the incidence and severity of the attacks appears to beincreasing. At the present time the number of pigs which are affectedannually by TGE is numbered in the millions and the economic lossresulting to the farmer from the ravages of the disease is severe.

The mechanism of transmission of TGE has not yet been fully determined,although birds, rodents, and wild camivores are suspected to becarriers. Isolation of infected pigs from all other animal life hasproved to be ineffective in controlling transmission of the disease,possibly indicating that transmission occurs through human contact orthrough aerial means.

All attempts to prevent or control the transmission of TGE to date havebeen failures or insufficiently effective for practical use. Among themethods which have been tried for controlling the disease, in theabsence of any other effective method, has been the feeding by farmersof virulent TGE virus to pregnant sows in the hope that sufiicientantibodies would be produced in their milk for conferring some immunityto the nursing piglets. Although this method does in fact partiallycontrol the disease, it is unsatisfactory for the following reasons. Theinfected sow is more likely to abort her litter or to produce aninadequate quantity of milk in which the antibody titers may be low. Inaddition, the infection may be accidentally transferred to otherpreviously non-infected herds or persistent, or enzooic, TGE could beestablished within the herd, resulting in continuous disruption in afarrowing program. Clearly, the practice of infecting pregnant sows withvirulent TGE virus is not only inadequate as a method for controllingthe transmission of the disease among baby pigs, but is also dangerousand ill-advised.

Other attempts to control the transmission of TGE have involved the useof modified live virus, allegedly nonvirulent in nature, or attenuatedTGE virus for inoculating pregnant sows, again with the expectation thatantibodies would be transferred to the offspring through the sows milk.Such methods have similarly been found to be ineffective in controllingthe disease. Since baby pigs are immunologically immature, they cannotproduce protective antibodies in response to a vaccine administereddirectly. Accordingly, since TGE can be fatal to newborn pigs within aperiod of less than 24 hours after exposure, the piglets are susceptibleto TGE in the event that the pregnant sow has not produced suflicientantibodies or sufiicient milk for protection of the litter of baby pigs.In addition, any method which relies solely on vaccination of thepregnant sow for conferring immunity to the litter of piglets requiresthat such vaccinations be performed continuously even when there is noevidence of an outbreak of TGE. Protection of baby pigs from anunvaccinated sow has been impossible until the present invention.

In its broadest aspect, the present invention provides a method ofadministering to an animal any villiarly active biological, i.e., anybiological which can be absorbed by the villi of the small intestine toproduce a desired effect, by orally administering to the animal saidbiological in the form of enterically coated particles having acontrolled maximum size. The method of the invention is particularlyapplicable to biologicals which are sensitive to the gastric secretionsof the animal or susceptible to degradation by body heat. Thebiologicals used in the invention are prepared in a dosage formcomprising solid particles consisting of the biological alone or of aphysiologically acceptable solid carrier on or in which the biologicalis deposited, the particles having a critical maximum size and anenteric coating to protect against degradation by contact with thegastric secretions of the animal. We have found that by limiting thesize of the particles, passage thereof through the pyloric valve .of theanimal into the small intestine occurs within such a short time thatconventional enteric coatings are adequate to prevent degradation by thegastric juices or destruction of effectiveness which might result fromthe animals body heat in the event the particles remain in the animalsstomach for an excessive period of time.

The term biological, as used herein, refers to substances which areadministered to animals for the effect which they have on the processesproducing immunity in the animal, such as vaccines, viruses, toxins,antigens, antibodies, and the like.

The invention stems in part from our discovery that the time ofretention of particles in the stomach of an animal, before the particlespass through the pyloric valve into the animals intestine, is dependenton the size of the particles. In any given species of animal, there is acritical maximum particle size above which the retention time in thestomach rapidly increases and below which the retention time is minimal.For example, in the pig we have determined that whereas pellets whichare approximately spherical in shape and have a diameter less than about2.0 millimeters, and preferably less than about 1.5 millimeters, willpass from the stomach through the pyloric valve into the intestinewithin reasonably short periods, on the order of 1.5 hours or less,larger pellets remain in thestomach for periods of time which increasesharply with increasing size; for example, pellets having a diameter of3 mm. may remain in the stomach of a pig for as long as two days. Wehave further determined that the enteric coatings conventionally used tocoat tablets provide in viva periods of protection which are much lessthan those expected from tests of the same tablets in vitro in simulatedgastric juices. Accordingly, in order to assure the potency of entericcoated villiarly administered biological materials which are sensitiveto heat or gastric secretions, it is essential that the maximum size ofthe particles used be limited, in accordance with the invention.

Following the method of the invention, we have found that it"is possibleto immunize both adult sows as well as newborn pigs against the ravagesof TGE by adminisration thereto of a dosage form of attenuated TGE viruscomprising micropellets, i.e., pellets having a diameter less than 2.0millimeters and preferably less than 1.5 millimeters, the' pelletscontaining a dried attenuated TGE virus vaccine produced in accordancewith conventional procedures. The micropellets are coated with aconventional enteric coating suitable for protecting the micropellets inthe stomach of the animal for a period of at least about two hours,within which time most of the pellets will have passed through theanimals pyloric valve into the small intestine, where the entericcoating rapidly dissolves and the vaccine becomes available forabsorption by the villi in the small intestine. When administered toadult swine in this manner, the vaccine causes the production ofantibodies within the animals system which effectively protect theanimal against infection by TGE. In the case of newborn pigs, which areimmunologically immature, the mechanism of protection appears to dependon interference by the vaccine with absorption and replication of TGEvirus in the animals villi, rather than on a true immunologicalreaction. In any event, and regardless of whether this hypothesis iscorrect, the fact remains that newborn pigs can be effectively protectedagainst TGE by orally administering to them enterically coatedmicroparticles of attenuated TGE virus vaccine in accordance with theinvention.

The specific method used to administer the enterically coatedmicroparticles of TGE to swine is not critical. It is important onlythat the individual particles be free to pass separately through thepyloric valve. Accordingly, the enteric coated microparticles can bemixed with the animals food in a concentration sufiicient to insure thatthe animal will have ingested a sufficient quantity of the vaccine toinsure protection. Thus, for baby pigs, the microparticles may be mixedwith wet baby food or viscous liquid feeds commonly used for piglets.For somewhat older pigs, the microparticles can be mixed in the startingmash and with still older pigs, the particles can be incorporated in thedry foods fed to the animals. Since, however, mixing the particles withthe animals food involves some uncertainty as to the amount of foodactually ingested, it is preferred that a known quantity of themicroparticles be fed directly into the stomach of the animals, as bythe use of a balling gun. In such cine is given, although this is notessential since we have found that the microparticles rapidly passthrough the pyloric valve even when the stomach is full of food.

Regardless of the specific methods used to administer the microcapsulesof the invention to swine, it is preferred that the dosage be inaccordance with the sample schedule below, in order to afford adequateprotection against TGE. Since viral strain may vary in liter, the exactdosage will vary with the particular biological in use. In general,however, the dosage increases with the age of the pig as follows.

For maximum protection, it is preferred that the above dosage berepeated after an interval of about two weeks.

The critical nature of the size of the particles on their retention timein a pigs stomach is demonstrated by the data of Table I. These datawere developed from tests in which macrocapsules of various volumes(0.5-20 ml.) containing steel pellets, 3 mm. in diameter, wereadministered intact to animals of various sizes. Radiographs of theanimals were taken at 15 minutes intervals, of both the right lateraland either the dorsal or ventral view, usually the latter to determinewhether the capsule had dissolved, and if so, whether the pellets werestill in the stomach. In each case it was found that the enteric coatedcapsule dissolved within the stomach in less than 2.5 hours and that thesteel pellets released when the capsules dissolved were retained in thestomach for five hours to two days. The enteric coatings used in thiswork were shellac and cellulose acetate phthalate (CAP), a commercialproduct sold especially for use as an enteric coating, which is made byreacting a partial acetate ester of cellulose with phthalic anhydride.The results set out in Table I demonstrate that the enteric coatingswere ineffective in protecting a capsule against dissolution in a pigsstomach for more than about 2 /2 hours at most and in many cases asubstantially less time. It was also found that pellets of 3 mm.diameter were retained in the stomach for a minimum of 5 hours and amaximum of 48 hours, regardless of whether the stomach was in an emptyor full condition when the capsules were administered.

TABLE I.PASSAGE OF ENTERIC COATED MACROCAPSULES THROUGH PIG STOMACHReten- Weight Dissolution tion of Sto h T 1 {me swine, mac me, n s om-Basic preparation Coating Filling lbs. condition hrs. Place ach, hrs 20ml. capsule 10 01:8. shellac Pellets, 3 mm. diam 2% Stomaeh-... 12 D dodo 45 2 do 10 2 m1, capsule rln fin 45 2 12 Do do do 30 1% 10 0.5 m1. cp do do 45 1. 5 12 Do do 45 1. 8 10 Do 6 ets. GAP .-do 35 1.25 8 Dn (in(in 25 1 25 9 Do do do 25 1. 25 8 D do do 45 1. 5 9 Do 10 cts. CAP -do60 1.5 5 Do do do 60 1. 6 7 D do do 350 1. 5 48 D do do 350 1. 5 36 D dodo 400 1. 5 36 Do do do 400 1. 6 24 Cellulose acetate phthalate.

method, there can be employed quick dissolving gelatin capsules or thelike containing a known amount of enterically coated microparticles. Thecapsules quicklydissolve within the animals stomach, releasing theindividual enteric coated microparticles for passage through the animalspyloric valve into the small intestine. Since it is possible that thecontents of the stomach might interfere with the passage through thepyloric valve, it is preferred that the animals stomach be empty whenthe vac- Similar enteric coated macrocapsules were also fed to guineapigs and opossums. It was found that the enteric coating preventeddissolution of the capsule for 3 /2 to 4 /2 hours in the stomach of theopossum, but opened within an hour in the stomach of the guinea pig,indicating that CAP as an enteric coating is more successful in thegastric juices of the opossum than in those of swine or guinea pigs. Theirregularity of the dissolution time of such enteric coatedmacrocapsules, however, gives rise to the possibility of heatdegradation of the capsule contents, even though the enteric coatingsremain intact. The use of enteric coated capsules of relatively largesize is therefore not a reliable method of delivering a heatsensitivebiological product to the duodenum of an animal.

The difiiculties evidenced by the data of Table I are obviated by themethod of the invention employing enteric coated microcapsules whichreadily pass out of the stomach of the animal into the small intestinebefore degradation due to gastric juices or heat degradation can occur.We have found that the shape of the microcapsule has some effect on thespeed of passage through the pyloric valve, with spherical shapespassing with the least difiiculty. Accordingly, microparticles ofapproximately sperical configuration are preferred for use in theinvention.

The speed with which enteric coated microcapsules pass through thestomach of a pig into its small intestine is demonstrated by the data ofTable H. In each case the animal was fed a quick dissolving gelatincapsule containing microparticles of barium sulfate which had beenenterically coated with CAP. In the times noted in the table 3 TABLEIL-PASSAGE OF MICRO-ENCAPSULATED PELLETS THROUGH PIG STOMACH INTO SMALLINTESTINE Time before Weight dissolu- Place Coatof pig, tion, of dis-Basie preparation ing Filling bs. min. solution Micro-encapsulated CAPBarium 30 Small inpellets, 1.5 mm. sulfate. testine.

axim

Do. 23 a 0. 30 Do. 45 D0.

The use of the method of the invention in protecting baby pigs byvaccinating the pregnant sow is illustrated by the data of Table III. Inthis work, pregnant sows were vaccinated one month preparturition withan attenuated TGE virus in the form of enterically coated micropellets(less than 1.5 mm. in diameter) contained in a rapidly dissolvingcapsule. The micropellets comprised sugar pellets of appropriate size,onto which was absorbed a TGE virus which had been attenuated bystandard techniques of serial passage through a tissue culture mediumfollowed by freeze-drying in a vacuum. In Test 3, the sow was also givenintramammary injections with attenuated virus, while in Test 4, apathogenic form of TGE virus (2-1) was used. As controls, there wereused an unvaccinated sow and a group of one-day-old pigs. In each test,the piglets born to the vaccinated sow were challenged with 2-1 virus.The results indicate that the vaccination of the pregnant sows gave goodprotection, although a few of the baby pigs showed mild signs ofdisease. When the oral vaccination was coupled with intramammaryinjection, the baby pigs were afforded complete protection against asevere challenge.

TABLE IIL-VACCINATION OF PREGNANT SOWS WITH TGE VIRUS IN ENTERIO COATEDMICROPELLETS Number baby Num- Numpigs her her Percent Test Vaccinechalpigs pigs surnumber Subject type 1 lenged 3 sick died vival 1Pregnant M-l08, 10 3 0 sow. attenu- 7 7 7 0 g Dam un- 3 3 3 0 one day 0d. vacclnated.

l Quantity of vaccine was about 10 TC IDw, administered in rapiddissolving capsule.

Each mammary gland also injected with 5X106 TC IDso o! M-108 attenuatedvirus.

3 Challenge dose was 10 pig LDm of 2-1 virus. 4 Control. 5 Challengedose was contained in enteric coated micropellets.

Vaccination of a pregnant sow, as practiced in the work reported inTable HI, however, requires that the pregnant sow be vaccinated at atime sufficiently before parturition to permit the production ofantibodies. Since it cannot be predicted whether an active attack of TGEwill be present in the herd at the time of parturition, every pregnantsow must be vaccinated routinely in order to aiford protection to thebaby pigs.

TABLE 1V.COMPARISON OF INTERFERENCE BY ATTENUATED P408 TGE VIRUS INLIQUID FORM AND ENTERIC COATED P408 VIRUS TO CHALLENGE WITH PATHO GENICTGE VIRUS IN BABY PIGS Challenge 3 Protective inoculum Quan- Time, tity,v Quanhrs. pig in- Signs Age of Numtity, after fective observed, Testpigs, ber 10 TC 1nocdoses hrs. after number days used Form IDso ulum(PID) challenge Results TABLE IV-COMPARISON OF INTERFERENCE BYATTENUATED P-108 TGE VIRUS IN LIQUID FORM AND ENTERIC COATED P-l08 VIRUSTO CHALLENGE WITH PATHOGENIC. TGE VIRUS IN BABY PIGS Challenge 3Protective inoculum Quan- Time, fity, Quanhrs. pig in- Signs Age ofNmnafter iective observed, pigs, inocdoses hrs. after days ulum (PID)challenge Results 2 24 100 28 Diarrhea, vomiting; death. 2 24 100 30 D0.2 24 100 48 Soft feces; survived. 2 d 24 100 None Survived. 2 q 48 10030 Severe diarrhea; k5 survived. 2 1 g i 32 r i d: h

omi in arr ea death. 3 Liquid 1 5 4s 10, 000 48 Do. 3 do 30 48 10,00048,72 Diarrhea; death. 3 2 Entenc coated 2 48 10,000 1 pig, slightdiarrhea; second pig, none; both survived. 3 2 .do 4 48 10,000 NoneSurvived. 3 2 Placebo 48 10,000 18 Vomiting, diarrhea; death.

1 Fluid from P.K. cell culture titering about 10 TC ID per ml. 2Pelleted microparticles in a capsule (non-coated) titering about 10 TCIDto per capsule. Challenge was with 2-1 lyophilized and reconstitutedvirus.

The method of the invention lessens the need for routine vaccination inthis manner, permitting baby pigs to be protected at birth in the eventthat TGE is present in the herd at the time. Although the most completeprotection is afforded by vaccination of both the soW (includingintramammary injection) as well as the baby pigs, treatment of the babypigs alone is also elfective, as shown in Table IV, which shows theresults obtained by inoculating baby pigs born to unvaccinated sows.Table IV also compares the results obtained by feeding a liquidinoculum, i.e., not enteric coated, of an attenuated TGE virus withthose obtained with enteric coated microparticles, in accordance withthe invention. As can be seen from the data of the table, the uncoatedattenuated virus was inelfective against both moderate (Tests 1, 2, aswell as severe (Tests 8, 9) challenges. By contrast, the enteric coatedinoculum (Tests 3, 4, 6 10 and 11) in each case protected the baby pigs,in most cases without even any signs of sickness.

The attenuated oral vaccine contemplated for use in the presentinvention can be produced by replication of the TGE virus by standardtechniques. If it is to be produced from an animal source, the animalmust first be infected with live, active TGE virus. Dogs, pigs, or othersubjects known to be susceptible may be used. The infected tissues,principally the small intestine, are subjected to trituration,homogenization, extraction with an appropriate bulfered solution, andcentrifugation in order to recover the virus. The extracted virus nextis purified by means of sucrose density gradient centrifugation. Thedose of virus is then standardized to an infective dose of 50% (ID50).

The purified, standardized virus is then lyophilized (freeze-dried) Thematerial is transferred to shallow pans and deep frozen, and moisture iseliminated at conditions below the eutectic point of water. Thelyophilized material must then be stored in a cool dry, place, afterwhich the bulk lyophilized material is reduced to a fine powder bycrushing in a hammer or knife mill. To keep the temperature down, solidcarbon dioxide may be milled together with it.

Pelletizing is the next step. The powered attenuated virus is coatedonto processed nonpareil (sugar) seeds (or other physiologicallyacceptable solid carriers) of the appropriate diameters, usingacceptable binders and film barrier formers such as arsenic-free shellacor cellulose acetate phthallate (CAP) or others in appropriate solventsand drying agents. At all times, care should be taken not to expose thevirus to high temperatures.

Although TGE, which primarily affects swine, has been emphasized in thisdisclosure, the present invention is not intended to be limited to anyparticular biological, disease, or animal. As modifications will beobvious to those skilled in the art, the foregoing descriptions havebeen given for clearness of understanding only, and no unnecessarylimitations should be understood therefrom.

We claim:

1. The method of vaccinating a pig against TGE, which pig if adult orimmunologically mature is capable on vaccination of producing antibodieswhich effectively protect the animal against infection by TGE and ifnewborn or otherwise immunologically immature is capable of absorbingand replicating viruses in the villi to interfere with TGE virus, whichmethod comprises perorally introducing into the stomach of said pigattenuated TGE virus vaccine in the form of approximately sphericalmicroparticles having a maximum diameter less than about 2.0 mm., saidvaccine being subject to degradation by contact with the gastricsecretions of said pig, said particles having an enteric coating whichis relatively insoluble in the stomach at said pig but which dissolvesrapidly in the intestine, whereby said particles pass from the stomachthrough the pyloric valve into the intestine before said enteric coatingis destroyed by contact with gastric secretions, the dosage of saidvaccine being about 8 10 to 10 T.C. ID for immature pigs and about 10 to10 T.C. ID for mature pigs.

2. The method of claim 1 wherein said microparticles are contained in adosage form comprising a container which dissolves readily in thestomach of said pig, releasing said enterically coated microparticlesfor passage through the pyloric valve of said pig.

3. The method of claim 1 wherein said particles have a maximum diameterless than about 1.5 mm.

4. The method of claim 3 wherein said vaccine is administered at adosage rate in accordance with the following schedule:

5. The method of claim 1 wherein said pig is a pregnant sow, and saidvaccination occurs a suflicient period of time before parturition tocause the production of antibodies effective against TGE, whichantibodies are transmitted via the sows milk to the suckling pigletsafter parturition.

6. A biological product for oral administration to a pig for protectionagainst TGE comprising TGE virus vac-. cine in the form of approximatelyspherical microparticles, said vaccine being subject to degradation bycontact with the gastric secretions of said pig, with an enteric coatingcovering each particle of said vaccine, said microparticles having amaximum diameter less than about 2.0 mm., whereby they will pass fromthe stomach through the pyloric valve into the intestine of said pigbefore said enteric coating dissolves in said gastric secretions.

7. The biological product of claim 6 wherein said microparticles have amaximum diameter less than about 8. A biological product in dosage formfor oral administration to swine for protection against TGE comprisingTGE attenuated virus vaccine in solid micropar ticulate form, eachparticle of said vaccine having an enteric coating, said particles beingapproximately spherical and having a maximum diameter less than about1.5 mm., and encapsulation means for holding said microparticles indosage form, said dosage form containing about 10 to 10 T.C. ID of saidvaccine, said encapsulation means being readily soluble in contact withthe gastric secretions of the animal to which it is administered, saidenteric coating being eifective to prevent degradation of said vaccineby said gastric secretions for the time said particles remain in thestomach of said animal, but being readily soluble in the intestinalsecretions thereof after said microparticles have passed through thepyloric valve.

9. The vaccine of claim 7 in which said enteric coating comprisescellulose acetate phthalate.

10. The vaccine of claim 9 in which said vaccine is deposited on or in aphysiologically acceptable solid particulate carrier.

References Cited UNITED STATES PATENTS 3,081,233 3/1963 Enz et al.424--33 2,957,804 10/1960 Shuyler 424--17 3,519,710 7/1970 Bass 424-893,458,621 7/1969 Tint 424-35 3,072,528 1/ 1963 Kludas et al. 424-933,376,199 4/1968 Coles et al. 424-89 3,317,393 5/1967 Chanock et al.424-89 X 2,369,218 2/ 1945 Dick et a1 424-93 X 3,541,203 11/1970 Fogleet al. 424--93 X 2,946,724 7/ 1960 Valentine 424-89 3,127,318 3/ 1964Eversole et al 424-89 3,585,108 6/1971 Welter 424-89 SHEP K. ROSE,Primary Examiner US. Cl. X.R.

gzgy UNITED STATES PATENT OFFICE 7 CERTIFICATE OF CORRECTION Patent No.3,323,223 I v Dated y 1974 Inventor(s) Deam H. Ferris and Angel SArambulo It is certified that error appears in the above-identifiedpatent and that said Letters Patent are hereby corrected as shown below:

Column 4, line 8, "strain" should be strains-; Column 5, line-23,"sperical" should be -spherical-; Columns 5 and 6, line. 48', Table IVheading is duplicated; Column 7, line 17, after "6" insert a commaColumn 7, line 37, after "cool" insert a comma Column 7, Iine 4"powered" should be --powdered-.-.

Signed and sealed this 8th da of October 1974.

(SEAL) Attest:

MCCOY M. GIBSON JR. C. MARSHALL DANN Attesting Officer Commissioner ofPatents

